JP7576024B2 - Histochemical and cytochemical methods for detecting NTRK fusion proteins - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62/731,032, filed Sep. 13, 2018, entitled "HISTOCHEMICAL AND CYTOCHEMICAL METHODS FOR DETECTING NTRK FUSION PROTEINS," the contents of which are incorporated herein by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING This application incorporates by reference the sequence listing submitted herewith in computer readable form with file name "P35023-WO PCT fling sequence listing" (21,260 bytes), created on September 9, 2019.

The present disclosure relates, inter alia, to methods for the histochemical and cytochemical detection of fusion proteins involving NTRK gene products, materials, kits, and systems useful in such methods, and products resulting from the practice of such methods.

Neurotrophic tyrosine receptor kinases (NTRK1, NTRK2, and NTRK3) are a family of genes encoding receptor tyrosine kinase proteins (TRKA, TRKB, and TRKC) that play a role in the development and maturation of the central and peripheral nervous systems (Barbacid I, Barbacid II, Lemmon & Schlessinger, and Klein). In cancer, the intact kinase domain of one of the three NTRK genes can be fused to various upstream partners (ETV6, LMNA, TPM3, etc.), replacing the ligand-binding domain of the TRK protein with a structural motif that promotes dimerization (Eide, Luberg, & Vaishnavi). This leads to constitutive activation of TRK signaling and unrestrained proliferation due to expression of the fusion protein.

NTRK gene fusions are characteristic of certain rare tumors such as infantile fibrosarcoma, congenital mesodermal nephroma, secretory breast cancer, and secretory carcinoma of the salivary glands (MASC) (Argani, Bishop, Bourgeois, Rubin, & Tognon). Conversely, NTRK gene fusions occur rarely in adult and pediatric solid tumors, including but not limited to appendix cancer, breast cancer, cholangiocarcinoma, colorectal cancer (CRC), GIST, lung cancer, melanoma, pancreatic cancer, thyroid cancer, and various sarcomas (DeBraud, Brzezianska, Fernandez-Cuesta, Leeman-Neill, & Ross). Identifying NTRK gene fusions is of clinical importance because small molecule inhibitors currently in development for the treatment of solid tumors with NTRK gene fusions are available (Vaishnavi & De Braud).

To date, there is no gold standard for detecting NTRK fusions in tumors due to inconsistent specificity and sensitivity between different methodologies. Current detection methods include fluorescent in situ hybridization (FISH), in situ hybridization (ISH), next generation sequencing (NGS), and immunohistochemistry (IHC). Optimization and enumeration/interpretation of FISH and ISH assays can be difficult. NGS is highly specific but lacks sensitivity. In contrast, IHC can be highly sensitive but detects protein expression rather than the actual presence of the fusion. In addition, due to the endogenous presence of wild-type TRK protein in some tumors (i.e. neuroendocrine tumors and GIST), optimization of IHC assays for the fusion product, and in turn, development of specific scoring algorithms to select for this fusion product, can be challenging.

The present disclosure relates to a method for identifying non-neuroendocrine tumors driven by TRK fusion proteins.

In one embodiment, a method is provided for detecting a fusion protein of TrkA, TrkB, or TrkC in a non-neuroendocrine tumor sample, the method comprising: affinity histochemically staining the sample with a biomarker-specific reagent that specifically binds to one or more of an amino acid sequence comprising, consisting essentially of, or consisting of residues 363-760 of SEQ ID NO:1; an amino acid sequence comprising, consisting essentially of, or consisting of residues 646-838 of SEQ ID NO:2; or an amino acid sequence comprising, consisting essentially of, or consisting of residues 718-839 of SEQ ID NO:3; detecting a staining pattern in the sample; and scoring the sample as positive for a fusion protein involving TrkA, TrkB, or TrkC if the sample has a cytoplasmic and/or membranous staining pattern and has equal to or greater than a first threshold percentage of tumor cells that stain above a predetermined specific staining intensity, or if the sample has a nuclear staining pattern and equal to or greater than a second threshold percentage of cells within a threshold tumor cell area that stain specifically at any intensity.

In one embodiment, a method is provided for detecting a fusion protein of TrkA, TrkB, or TrkC in a non-neuroendocrine tumor sample, the method comprising affinity histochemically staining the sample with a biomarker-specific reagent that specifically binds to an amino acid sequence comprising, consisting essentially of, or consisting of residues 363-760 of SEQ ID NO:1; an amino acid sequence comprising, consisting essentially of, or consisting of residues 646-838 of SEQ ID NO:2; and an amino acid sequence comprising, consisting essentially of, or consisting of residues 718-839 of SEQ ID NO:3; detecting a staining pattern in the sample; and scoring the sample as positive for a fusion protein involving TrkA, TrkB, or TrkC if the sample has a cytoplasmic and/or membranous staining pattern and has equal to or greater than a first threshold percentage of tumor cells that stain above a predetermined specific staining intensity, or if the sample has a nuclear staining pattern and equal to or greater than a second threshold percentage of cells within a threshold tumor cell area that stain specifically at any intensity.

In one embodiment, a method is provided for detecting a fusion protein of TrkA, TrkB, or TrkC in a non-neuroendocrine tumor sample, the method comprising affinity histochemically staining the sample with a biomarker-specific reagent that specifically binds to an amino acid sequence consisting of amino acids 816-838 of SEQ ID NO:2, detecting a staining pattern in the sample, and scoring the sample as positive for a fusion protein involving TrkA, TrkB, or TrkC if the sample has a cytoplasmic and/or membranous staining pattern and has equal to or greater than a first threshold percentage of tumor cells that stain above a predetermined specific staining intensity, or if the sample has a nuclear staining pattern and equal to or greater than a second threshold percentage of cells within a threshold tumor cell area that are specifically stained at any intensity.

In one embodiment, a method is provided for detecting an NTRK rearrangement in a non-neuroendocrine tumor sample, the method comprising detecting the presence of a Trk fusion protein in the sample according to the methods described herein, and if the sample scores positive for a fusion protein involving TrkA, TrkB, or TrkC, confirming the presence of an NTRK rearrangement by screening the sample by sequencing, reverse transcription polymerase chain reaction, or in situ hybridization.

In one embodiment, a method is provided for selecting a patient to receive a Trk-directed therapy, the method comprising detecting the presence of a Trk fusion protein or an NTRK rearrangement in a non-neuroendocrine tumor sample according to the methods described herein, and selecting the patient to receive the therapy if the sample scores as positive for a fusion protein involving TrkA, TrkB, or TrkC, or if an NTRK rearrangement is detected.

In one embodiment, a method of staining a sample for the presence or absence of TrkA, TrkB, or TrkC, and fusion proteins containing their kinase domains, is also provided, the method comprising (a) subjecting the sample to a heat-induced epitope retrieval process, and (b) contacting the sample with a set of biomarker-specific and detection reagents to deposit a bright field dye proximate any biomarker-specific reagents bound to the sample, wherein the biomarker-specific reagents specifically bind to one or more of an amino acid sequence comprising, consisting essentially of, or consisting of residues 363-760 of SEQ ID NO:1; an amino acid sequence comprising, consisting essentially of, or consisting of residues 646-838 of SEQ ID NO:2; or an amino acid sequence comprising, consisting essentially of, or consisting of residues 718-839 of SEQ ID NO:3.

In one embodiment, a method of staining a sample for the presence or absence of TrkA, TrkB, or TrkC, and fusion proteins containing their kinase domains, is also provided, the method comprising: (a) subjecting the sample to a heat-induced epitope retrieval process; and (b) contacting the sample with a set of biomarker-specific and detection reagents to deposit a brightfield dye near any biomarker-specific reagent bound to the sample, where the biomarker-specific reagent is a primary antibody that specifically binds to one or more of: an amino acid sequence comprising, consisting essentially of, or consisting of residues 363-760 of SEQ ID NO:1; an amino acid sequence comprising, consisting essentially of, or consisting of residues 646-838 of SEQ ID NO:2; or an amino acid sequence comprising, consisting essentially of, or consisting of residues 718-839 of SEQ ID NO:3; and the set of detection reagents comprises a secondary antibody immunoreactive with the primary antibody, a tertiary antibody immunoreactive with the secondary antibody conjugated to an enzyme, and a set of reagents reactive with an enzyme that result in the deposition of the brightfield dye in the sample.

In one embodiment, a method of staining a sample for the presence or absence of TrkA, TrkB, or TrkC, and fusion proteins containing their kinase domains, is also provided, the method comprising: (a) subjecting the sample to a heat-induced epitope retrieval process; and (b) contacting the sample with a set of biomarker-specific and detection reagents to deposit a brightfield dye near any biomarker-specific reagent bound to the sample, where the biomarker-specific reagent is a primary antibody that specifically binds to each of: an amino acid sequence comprising, consisting essentially of, or consisting of residues 363-760 of SEQ ID NO:1; an amino acid sequence comprising, consisting essentially of, or consisting of residues 646-838 of SEQ ID NO:2; or an amino acid sequence comprising, consisting essentially of, or consisting of residues 718-839 of SEQ ID NO:3; and the set of detection reagents comprises a secondary antibody immunoreactive with the primary antibody, a tertiary antibody immunoreactive with the secondary antibody conjugated to an enzyme, and a set of enzyme-reactive reagents that result in the deposition of the brightfield dye in the sample.

Other embodiments will become apparent from the disclosure below.

Sequence alignment between exemplary conserved portions of TrkA (amino acid residues 363-760 of SEQ ID NO:1), TrkB (amino acid residues 646-838 of SEQ ID NO:2) and TrkC (amino acid residues 718-839 of SEQ ID NO:3). Sequence alignments were generated using the Kalign Multiple Sequence Alignment tool by EMBL-EBI (https://www.ebi.ac.uk/Tools/msa/kalign/). Default settings were used, including ClustalW output format, a gap open penalty of 11, a gap extension penalty of 0.85, a terminal gap penalty of 0.45, and a bonus score of 0. IHC Results Trk Fusions in Multiple Tumor Types Orthogonal testing methods were used to assess the presence of Trk fusions. IHC results in Trk fusions in multiple tumor types. Orthogonal testing methods were used to assess the absence of Trk fusions. Staining distribution of tumor types of ISH-confirmed fusion-positive cases in the dataset by percentage of tumor cell staining (0-100%) and intensity of staining (0-3+). Circles represent fusion-negative cases and triangles represent fusion-positive cases. Diamonds indicate cases where fusion was detected by ISH but a fixed gradient was observed. Patterns represent different tumor histologies. Figures 5a and 5b show the staining distribution of neuroendocrine tumors where no fusion-positive cases were detected. Staining distribution of tumor types of ISH-confirmed fusion-negative cases within the dataset by percentage of tumor cell staining (0-100%) and intensity of staining (0-3+). Subcategorization of solid tumors based on IHC and ISH testing. Examples of staining pattern localization differences obtained with the staining methods described herein: (A) Cytoplasmic/membrane staining pattern in a colorectal tumor determined to have an NTRK1 rearrangement by break-apart in situ hybridization assay and confirmed to have an NTRK1-TPM3 fusion by next generation sequencing. (B) Cytoplasmic staining pattern in a colorectal tumor determined to have an NTRK1 rearrangement by break-apart in situ hybridization assay and confirmed to have an NTRK1-EML4 fusion by next generation sequencing. (C) Nuclear/cytoplasmic staining pattern in a mammary analogue secretory carcinoma (MASC) determined to have an NTRK3 rearrangement by break-apart in situ hybridization assay and confirmed to have an NTRK3-ETV6 fusion by next generation sequencing. An example of a fixed gradient within a tissue, and a demonstration that nuclear localization appears to be more affected by the gradient than cytoplasmic/membrane localization. The bottom row shows low magnification, and the top row shows high magnification of a subsection of the image immediately below.

I. Definitions Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.See, for example, Lackie, DICTIONARY OF CELL AND MOLECULAR BIOLOGY, Elsevier (4th ed. 2007); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989).The term "a" or "an" is intended to mean "one or more." The terms "comprise", "comprises" and "comprising" when preceding a list of steps or elements are intended to mean that the addition of further steps or elements is optional and is not excluded.

Antibody: The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

Antibody fragment: "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, bispecific antibodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

Biomarker: As used herein, the term "biomarker" is intended to refer to any molecule or group of molecules found in a biological sample that can be used to characterize the biological sample or the subject from which the biological sample is obtained. For example, a biomarker can be a molecule or group of molecules whose presence, absence, or relative abundance is:
- characteristics of particular cell or tissue types or conditions;
- a characteristic of a particular pathological condition or condition; or - an indication of the severity of the pathological condition, the likelihood of progression or regression of the pathological condition, and/or the likelihood that the pathological condition will respond to a particular treatment.

As another example, a biomarker can be a cell type or a microorganism (bacteria, mycobacteria, fungi, viruses, etc.), or a substitute molecule or group of such molecules.

Biomarker-specific reagent: A specific detection reagent, such as a primary antibody, that can bind directly and specifically to one or more biomarkers in a cell sample.

Cell Sample: As used herein, the term "cell sample" refers to any sample containing intact cells, such as a cell culture, a body fluid sample, or a surgical specimen taken for pathological, histological, or cytological interpretation.

Cytochemical detection: A process involving labeling a biomarker or other structure in a cytological sample with a biomarker-specific reagent and a detection reagent in a manner that allows for microscopic detection of the biomarker or other structure in the context of intact cells.

Cytological sample: As used herein, the term "cytological sample" refers to a cell sample that does not have cross-sectional spatial relationships in vivo (e.g., cell samples derived from blood samples, urine samples, sputum, etc.) or in which the cross-sectional spatial relationships have been at least partially destroyed (e.g., tissue smears, liquid-based cytology samples, fine needle aspirates, etc.).

Detection Reagent: A "detection reagent" is any reagent used to deposit a stain adjacent to a biomarker-specific reagent in a cell sample. Non-limiting examples include a biomarker-specific reagent (such as a primary antibody), a secondary detection reagent (such as a secondary antibody that can bind to a primary antibody), a tertiary detection reagent (such as a tertiary antibody that can bind to a secondary antibody), an enzyme directly or indirectly associated with a biomarker-specific reagent, chemicals reactive with such enzymes that result in the deposition of a fluorescent or chromogenic stain, wash reagents used between staining steps, etc.

Detectable moiety: A molecule or material capable of producing a detectable signal (visual, electronic, etc., or other) that indicates the presence (i.e., qualitative analysis) and/or concentration (i.e., quantitative analysis) of a detectable moiety deposited on a sample. The detectable signal may be produced by any known or yet to be discovered mechanism, including absorption, emission, and/or scattering of photons (including radio frequency, microwave frequency, infrared frequency, visible frequency, and ultraviolet frequency photons). The term "detectable moiety" includes chromogenic, fluorescent, phosphorescent, and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance to another to provide a detectable difference (by converting a colorless substance to a colored substance or vice versa, or by producing a precipitate or increasing the turbidity of the sample). In some examples, the detectable moiety is a fluorophore, which belongs to several common chemical classes, including coumarins, fluoresceins (or fluorescein derivatives and analogs), rhodamines, resorufins, luminophores, and cyanines. Additional examples of fluorescent molecules are described in Molecular Probes Handbook-A Guide to Fluorescent Probes and Labeling Technologies, Molecular Probes, Eugene, OR, ThermoFisher Scientific, 11th Edition. In other embodiments, the detectable moiety is diaminobenzidine (DAB), 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL), tetramethylrhodamine (DISCOVERY Purple), N,N'-biscarboxypentyl-5,5'-disulfonato-indo-dicarbocyanine (Cy5), and rhodamine 110 (rhodamine).

Histochemical detection: A process involving labeling a biomarker or other structure in a tissue sample with a biomarker-specific reagent and a detection reagent in a manner that allows for microscopic detection of the biomarker or other structure in relation to the cross-sectional relationships between the structures of the tissue sample.

Monoclonal antibody: An antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and/or bind the same epitope, with the exception of variant antibodies that may contain naturally occurring mutations or arise during the manufacture of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the nature of the antibody being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, or a combination thereof.

"Retained portion" shall mean any portion of the wild-type counterpart of an oncogenic fusion protein that is conserved in the oncogenic fusion protein.

Specific Binding: As used herein, the phrases "specific binding," "specifically binds to," or "specific for" refer to a measurable and reproducible interaction between a target and a specific detection reagent that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that specifically binds to a target is an antibody that binds to the target with higher affinity, avidity, more readily, and/or with longer duration than it binds to other targets. In one embodiment, the extent to which a specific detection reagent binds to an unrelated target is less than about 10% of the binding of an antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, a biomarker-specific reagent that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In another embodiment, specific binding can include, but does not require, exclusive binding.

Specific detection reagent: Any composition of matter capable of specifically binding to a target chemical structure in the context of a cell sample.

Stain: When used as a noun, the term "stain" shall refer to any substance that can be used to visualize specific molecules or structures in a cell sample for microscopic analysis, including bright field microscopy, fluorescence microscopy, electron microscopy, and the like. When used as a verb, the term "stain" shall refer to any process that results in the deposition of a colorant in a cell sample.

Subject: As used herein, the term "subject" or "individual" refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, horses, etc.), primates (e.g., humans, non-human primates such as monkeys, rabbits, rodents (e.g., mice, rats, etc.), and the like. In certain embodiments, the individual or subject is a human.

Test sample: A tumor sample obtained from a subject whose outcome is unknown at the time the sample is obtained.

Tissue Sample: As used herein, the term "tissue sample" refers to a sample of cells that preserves the cross-sectional spatial relationships between cells that were present within the subject from which the sample was obtained.

Tumor sample: A tissue sample obtained from a tumor.

II. Staining Method The present disclosure is based on affinity histochemical or cytochemical staining samples of non-neuroendocrine NTRK fusion proteins using a first biomarker-specific reagent that targets one or more retained portions of TrkA, TrkB, and TrkC. The biomarker-specific reagent is selected by identifying the breakpoint of the wild-type protein that results in the fusion protein and targeting the part of the wild-type protein that is on the side of the breakpoint that is retained in the fusion protein. Many resources are available for identifying the breakpoint of fusion proteins, such as the COSMIC database, which includes annotation of the breakpoint by genomic location and the first exon expressed in the resulting fusion protein. An exemplary wild-type protein sequence is shown in Table 1.

Exemplary breakpoints and consensus retained portions of fusion proteins retaining the C-terminus of TrkA, TrkB, or TrkC are shown in Table 2.

In one embodiment, the biomarker-specific reagent specifically binds to one or more retention portions of TrkA, TrkB, and TrkC. In one embodiment, the biomarker-specific reagent specifically binds to the retention portions of each of TrkA, TrkB, and TrkC. An alignment between exemplary retention portions of TrkA, TrkB, and TrkC is shown in FIG. 1. In another specific embodiment, the biomarker-specific reagent specifically binds to one or more of the following: (a) an amino acid sequence comprising, consisting essentially of, or consisting of residues 363-760 of SEQ ID NO:1; (b) an amino acid sequence comprising, consisting essentially of, or consisting of residues 646-838 of SEQ ID NO:2; or (c) an amino acid sequence comprising, consisting essentially of, or consisting of residues 718-839 of SEQ ID NO:3. In another particular embodiment, the biomarker-specific reagent specifically binds to: (a) an amino acid sequence comprising, consisting essentially of, or consisting of residues 363-760 of SEQ ID NO:1; (b) an amino acid sequence comprising, consisting essentially of, or consisting of residues 646-838 of SEQ ID NO:2; and (c) an amino acid sequence comprising, consisting essentially of, or consisting of residues 718-839 of SEQ ID NO:3.

The biomarker-specific reagent can be any type of entity useful for in situ detection (such as by histological or cytological staining methods) of a protein expressed by a cell sample. Exemplary biomarker-specific reagents include antibodies and antigen-binding fragments thereof, as well as antibodies and antigen-binding fragments thereof, such as ADNECTIN (10th FN3 fibronectin-based scaffold; Bristol-Myers-Squibb Co.), AFFIBODY (scaffold based on the Z domain of protein A from S. aureus; Affibody AB, Solna, Sweden), AVIMER (domain A/LDL receptor-based scaffold; Amgen, Thousand Oaks, Calif.), dAb (scaffold based on VH or VL antibody domains; GlaxoSmithKline PLC, Cambridge, UK), DARPin (ankyrin repeat protein-based scaffold; Molecular Partners AG, Zurich, Switzerland), ANTICALIN (lipocalin-based scaffold; Pieris AG, Fleising, Germany), NANOBODY (VHH (camelid Ig-based scaffold); Ablynx N/V, Ghent, Belgium), TRANS-BODY (transferrin-based scaffold; Pfizer Inc., New York, NY), SMIP (Emergent Biosolutions, Inc., Rockville, MD), and TETRANECTIN (C-type lectin domain (CTLD)-based scaffold), tetranectin; Borean Pharma A/S, Aarhus, Denmark). Descriptions of such engineered specific binding structures can be found in Wurch et al. , Development of Novel Protein Scaffolds as Alternatives to Whole Antibodies for Imaging and Therapy: Status on Discovery Research and Clinical Validation, Current Pharmaceutical Biotechnology, Vol. 9, pp. 502-509 (2008), the contents of which are incorporated by reference.

In certain embodiments, the biomarker-specific reagent is an antibody. In another particular embodiment, the antibody is a monoclonal antibody (such as a mouse monoclonal antibody or a rabbit monoclonal antibody). In another particular embodiment, the antibody specifically binds to one or more retained portions of TrkA, TrkB, or TrkC. In another particular embodiment, the antibody specifically binds to an epitope comprised in one or more of the following: (a) an amino acid sequence comprising, consisting essentially of, or consisting of residues 363-760 of SEQ ID NO:1; (b) an amino acid sequence comprising, consisting essentially of, or consisting of residues 646-838 of SEQ ID NO:2; or (c) an amino acid sequence comprising, consisting essentially of, or consisting of residues 718-839 of SEQ ID NO:3. In another specific embodiment, the antibody specifically binds to: (a) an amino acid sequence comprising, consisting essentially of, or consisting of residues 363-760 of SEQ ID NO: 1; (b) an amino acid sequence comprising, consisting essentially of, or consisting of residues 646-838 of SEQ ID NO: 2; and (c) an amino acid sequence comprising, consisting essentially of, or consisting of residues 718-839 of SEQ ID NO: 3. In one embodiment, the antibody is a commercially available antibody according to Table 3.

In a particular embodiment, the antibody is EPR17341.

Biomarker-specific reagents are used to histochemically or cytochemically affinity stain samples suspected of having NTRK fusion proteins. Affinity histochemical and cytochemical staining techniques typically involve contacting a sample, deposited on a slide or other solid support, with a biomarker-specific reagent under conditions sufficient to allow specific binding between the biomarker-specific reagent and the biomarker of interest. Binding of the biomarker-specific reagent to the biomarker facilitates deposition of a detectable moiety onto the sample near the location containing the biomarker. The detectable moiety can be used to identify and/or quantify the biomarker to which the biomarker-specific reagent is directed. The presence and/or relative amount of the target in the sample can then be detected by detecting the signal generated by the detectable moiety.

The staining process may be manual, automated, or a combination of manual and automated steps. In one embodiment, the staining process may be performed on an automated advanced staining platform. An automated advanced staining platform typically includes at least the following: reservoirs of various reagents used in the staining protocol, a reagent dispensing unit in fluid communication with the reservoirs for dispensing the reagents onto the slides, a waste removal system for removing used reagents and other waste from the slides, and a control system for coordinating the operation of the reagent dispensing unit and the waste removal system. In addition to performing the staining process, many automated slide stainers can also perform steps incidental to staining (or are compatible with another system that performs such auxiliary steps), including slide baking (for attachment of samples to the slides), dewaxing (also called deparaffinization), epitope inactivation, counterstaining, dehydration and clearing, and mounting. Prichard describes several specific examples and various features of automated IHC/ISH slide stainers, including the intelliPATH (Biocare Medical), WAVE (Celerus Diagnostics), DAKO OMNIS and DAKO AUTOSTAINER LINK 48 (Agilent Technologies), BENCHMARK (Ventana Medical Systems, Inc.), Leica BOND, and Lab Vision Autostainer (Thermo Scientific) automated slide stainers. is the assignee of several U.S. patents disclosing systems and methods for performing automated analyses, including U.S. Pat. Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901, and 6,943,029, and U.S. Published Patent Application Nos. 20030211630 and 20040052685, each of which is incorporated herein by reference in its entirety. Commercially available staining units usually operate on one of the following principles: (1) open individual slide staining, where the slide is positioned horizontally and the reagents are dispensed as a puddle on the surface of the slide containing the tissue sample (as implemented e.g. in the DAKO AUTOSTAINER Link 48 (Agilent Technologies) and intelliPATH (Biocare Medical) stainers); (2) liquid overlay technique, where the reagents are either covered by or dispensed through an inert fluid layer deposited on the sample (VENTANA). (3) capillary gap staining, in which a slide surface is placed close to another surface (such as another slide or cover plate) to create a narrow gap through which capillary forces draw liquid reagents to keep them in contact with the sample (such as the staining principle used in the DAKO TECHMATE, Leica BOND, and DAKO OMNIS stainers). Over several iterations of capillary gap staining, the liquid in the gap is not mixed (such as in the DAKO TECHMATE and Leica BOND). A variation of capillary gap staining, called dynamic gap staining, uses capillary forces to apply the sample to the slide and then translates parallel surfaces into one another to agitate and mix the reagents during incubation (such as the staining principle implemented in the DAKO OMNIS slide stainer (Agilent)). In translation gap staining, a translatable head is placed over the slide. The underside of the head is separated from the slide by a first gap that is small enough for a meniscus of liquid to form from the liquid on the slide during translation of the slide. A mixing extension having a lateral dimension smaller than the width of the slide extends from the underside of the translatable head to define a second gap smaller than the first gap between the mixing extension and the slide. During translation of the head, the lateral dimension of the mixing extension is sufficient to generate a lateral movement in the liquid on the slide, generally in a direction extending from the second gap to the first gap. See WO 2011/139978 A1. It has recently been proposed to use inkjet technology to deposit reagents on slides. See WO 2016-170008 A1. This list of staining techniques is not intended to be comprehensive, and fully or semi-automated systems for performing biomarker staining can be used.

The staining method is performed on a cell sample of the suspected tissue, including tissue samples and cytological samples. In some embodiments, the cell sample is obtained from a subject having or suspected of having a tumor. In some embodiments, the sample includes cells obtained directly from the tumor. In some embodiments, the tumor is a solid tumor, such as a carcinoma, lymphoma, or sarcoma. In one embodiment, the tumor is a non-neuroendocrine tumor. In one embodiment, the non-neuroendocrine tumor is a solid tumor of the salivary gland, thyroid, skin, breast, head and/or neck, lung, upper gastrointestinal tract (including esophagus and stomach), female reproductive system (including tumors of the uterus, fallopian tubes, and ovaries), lower gastrointestinal tract (including tumors of the colon, rectum, and anus), genitourinary, exocrine, endocrine, renal, or lymphocytic origin. In one embodiment, the subject has melanoma, salivary gland cancer, thyroid cancer, breast cancer, ovarian cancer, pancreatic cancer, head and neck cancer, lung cancer, esophageal cancer, gastric cancer (excluding gastrointestinal stromal tumors (GIST)), colorectal cancer (including cancer of the colon, rectum, and anus), prostate cancer, urothelial cancer, or lymphoma.

If tissue samples are used, the tissue samples are processed in a manner compatible with histochemical staining, including, for example, fixation, embedding in a wax matrix (such as paraffin), and sectioning (such as with a microtome). No specific processing steps are required by the present disclosure, so long as the sample obtained is compatible with histochemical staining of the sample with a set of biomarker-specific reagents. In certain embodiments, microtome sections of formalin-fixed, paraffin-embedded (FFPE) samples are used in the staining process. If cytological samples are used, the samples are fixed in formalin.

Depending on the biomarker-specific reagent being used and the sample being used, the sample may be subjected to an epitope retrieval process (also called antigen retrieval) prior to application of the biomarker-specific reagent. Exemplary epitope retrieval processes include: heat-induced epitope retrieval (HIER), which involves heating the sample in various buffers at different pH levels; protease-based epitope retrieval (PBER), in which the sample is digested with proteolytic enzymes prior to staining; and a combination of HIER and PBER. Various specific epitope retrieval processes have been reviewed by Shi et al., D'Amico et al., Yamashita et al., Vinod et al., and Warford et al., but these are not exhaustive. Whether epitope retrieval is performed and the particular form of epitope retrieval used will depend on the particular biomarker-specific reagent selected and may need to be empirically determined for each biomarker-specific reagent used.

Depending on the reagents and samples used, it may be desirable to block endogenous protein activity prior to adding biomarker-specific and/or detection reagents. For example, if the detection reagent relies on biotin and a biotin-binding protein, it may be necessary to block endogenous biotin using, for example, free, unlabeled biotin-binding protein. Similarly, many detection schemes rely on the activity of enzymes such as phosphatases and peroxidases, and require neutralization of endogenous enzymes with similar activity. Commercially available kits are available for such blocking processes, including, for example, Endogenous Biotin Blocking Kit (Cat. No. E21390, ThermoFisher Scientific), Endogenous Avidin/Biotin Blocking Kit (Cat. No. ab64212, Abcam, plc.), Endogenous Biotin Blocking Kit Cat. No. 760-050, Ventana Medical Systems, Inc., among others. ), hydrogen peroxide blocking reagent (catalog number ab64218, Abcam plc.), and peroxidase and alkaline phosphatase blocking reagent (code S2003, Agilent Technologies).

Prior to applying the biomarker-specific reagent to the sample, it may also be useful to block sites on the sample to which the biomarker-specific reagent may nonspecifically bind. Common blocking agents include eBioscience™ IHC/ICC Blocking Buffer-High Protein (Cat. No. 00-4952-54, ThermoFisher Scientific), eBioscience™ IHC/ICC Blocking Buffer-Low Protein (Cat. No. 00-4953-54, ThermoFisher Scientific), DISCOVERY Antibody Block (Cat. No. 760-4204, Ventana Medical Systems, Inc.), along with normal serum buffer, nonfat dry milk, BSA (bovine serum albumin), and gelatin, among others.

Washing steps may be performed after each of these pretreatment steps by applying one or more passes of a wash buffer. The wash buffer is typically a neutral buffered saline solution, which may contain small amounts of detergent. Exemplary wash buffers include, for example, phosphate buffered saline (PBS), PBS-Tween 20, Tris-buffered saline (TBS), TBS-Tween 20 (polysorbate 20), Tris-HCl, Tris-HC-Tween 20, phosphate buffer (PB), AP buffer, etc.

Once the sample is prepared for staining, a biomarker-specific reagent is applied to the sample and incubated for a time and under conditions sufficient to promote specific binding between the biomarker and the biomarker-specific reagent. A wash step may be performed by applying one or more passes of a wash buffer after incubating the sample with the biomarker-specific reagent. This removes unbound or non-specifically bound biomarker-specific reagent from the sample and reduces off-target and/or background staining.

Detection of biomarkers in a sample is accomplished by depositing a detectable moiety in close proximity to the biomarker-specific reagent bound to the sample. In some cases, the detectable moiety is directly bound to the biomarker-specific reagent, so that it is deposited on the sample when the biomarker-specific reagent binds to its target (commonly referred to as direct labeling). In other embodiments, deposition of the detectable moiety is accomplished by applying a set of detection reagents to the sample after application of the biomarker-specific reagent, where the detection reagent binds to or otherwise reacts with the biomarker-specific reagent in a manner that results in deposition of the detectable moiety (commonly referred to as indirect labeling).

In some embodiments where an indirect method is used, the detectable moiety is deposited via an enzymatic reaction localized to the biomarker-specific reagent. Enzymes suitable for such reactions are well known and include, but are not limited to, oxidoreductases, hydrolases, and peroxidases. Specific enzymes explicitly mentioned are horseradish peroxidase (HRP), alkaline phosphatase (AP), acid phosphatase, glucose oxidase, β-galactosidase, β-glucuronidase, and β-lactamase. The enzyme may be directly attached to the biomarker-specific reagent or may be indirectly associated with the biomarker-specific reagent via a labeled conjugate. As used herein, a "labeled conjugate" includes the following:
(a) a specific detection reagent; and (b) an enzyme bound to the specific detection reagent, which reacts with a chromogenic substrate, a signaling conjugate and/or an enzyme-reactive dye under appropriate reaction conditions to result in the production of a dye in situ and/or deposition of a dye on a tissue sample.

In non-limiting examples, the specific detection reagent of the label conjugate can be a secondary detection reagent (such as a species-specific secondary antibody bound to a primary antibody, an anti-hapten antibody bound to a hapten-conjugated primary antibody, or a biotin-binding protein bound to a biotinylated primary antibody), a tertiary detection reagent (such as a species-specific tertiary antibody bound to a secondary antibody, an anti-hapten antibody bound to a hapten-conjugated secondary antibody, or a binding protein bound to a biotinylated secondary antibody), or other such arrangements. Enzymes localized to the biomarker-specific reagents bound to the sample in this manner can be used in many schemes to deposit detectable moieties.

In some cases, the enzyme reacts with a chromogenic compound/chromogenic substrate. Specific non-limiting examples of chromogenic compounds/chromogenic substrates include 4-nitrophenyl phosphate (pNPP), Fast Red, bromochloroindolyl phosphate (BCIP), nitroblue tetrazolium (NBT), BCIP/NBT, Fast Red, AP Orange, AP Blue, tetramethylbenzidine (TMB), 2,2'-azino-di-[3-ethylbenzothiazoline sulfonate] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN), nitrophenyl-β-D-galactopyranoside (ONPG), Examples include o-phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal), methylumbelliferyl-β-D-galactopyranoside (MU-Gal), p-nitrophenyl-α-D-galactopyranoside (PNP), 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazole (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blue, and tetrazolium violet.

In some embodiments, enzymes can be used in metallographic detection schemes. Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate for the enzyme. In some embodiments, the substrate is converted by the enzyme to a redox-active agent, which reduces the metal ion, causing it to form a detectable precipitate. (See, e.g., U.S. Patent Application Serial No. 11/015,646, filed December 20, 2004; PCT Publication No. 2005/003777; and U.S. Patent Application Publication No. 2004/0265922; each of which is incorporated herein by reference in its entirety). Metallographic detection methods include using an oxidoreductase (such as horseradish peroxidase) in combination with a water-soluble metal ion, an oxidizing agent, and a reducing agent to form a detectable precipitate. (See, e.g., U.S. Patent No. 6,670,113, incorporated herein by reference in its entirety).

In some embodiments, enzymatic action occurs between the enzyme and the dye itself, and the reaction converts the dye from an unbound species to a species that is deposited on the sample. For example, reaction of DAB with a peroxidase (such as horseradish peroxidase) causes DAB to be oxidized and precipitated.

In yet other embodiments, the detectable moiety is deposited via a signaling conjugate that includes a latent reactive moiety configured to react with an enzyme to form a reactive species that can bind to the sample or other detection components. These reactive species can react with the sample proximal to their generation, i.e., near the enzyme, but rapidly convert to a non-reactive species such that the signaling conjugate is not deposited at a site distal to the site where the enzyme is deposited. Examples of latent reactive moieties include quinone methide (QM) analogs, such as those described in WO2015124703A1, and tyramide conjugates, such as those described in WO2012003476A2, each of which is incorporated herein by reference in its entirety. In some examples, the latent reactive moiety is directly conjugated to a dye, such as N,N'-biscarboxypentyl-5,5'-disulfonato-indo-dicarbocyanine (Cy5), 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL), tetramethylrhodamine (DISCO purple), and rhodamine 110 (rhodamine). In other examples, the latent reactive moiety is conjugated to one member of a specific binding pair and the dye is conjugated to the other member of the specific binding pair. In other examples, the latent reactive moiety is linked to one member of a specific binding pair and an enzyme is linked to the other member of the specific binding pair, where the enzyme (a) is reactive with a chromogenic substrate to result in the production of a dye, or (b) is reactive with a dye to result in the deposition of a dye (such as DAB). Examples of specific binding pairs include:

(1) biotin or a biotin derivative (such as desthiobiotin) linked to a potentially reactive moiety, and a biotin-binding entity (avidin, streptavidin, deglycosylated avidin (such as NEUTRAVIDIN), or a biotin-binding protein (such as CAPTAVIDIN) with a nitrated tyrosine at its biotin-binding site linked to a dye or enzyme reactive with a chromogenic substrate or reactive with a dye (e.g., peroxidase linked to a biotin-binding protein when the dye is DAB); and (2) a hapten linked to a potentially reactive moiety, and an anti-hapten antibody linked to a dye, or an enzyme reactive with a chromogenic substrate or reactive with a dye (e.g., peroxidase linked to a biotin-binding protein when the dye is DAB).

Specifically included are non-limiting examples of combinations of biomarker-specific reagents and detection reagents set forth in Table 4.

In certain embodiments, the biomarker-specific reagents and specific detection reagents shown in Table 4 are antibodies. As will be appreciated by one of skill in the art, the detection schemes for each of the biomarker-specific reagents may be the same or different.

Non-limiting examples of commercially available detection reagents or kits that contain detection reagents suitable for use in the methods of the present invention include: VENTANA ultraView Detection System (secondary antibodies conjugated to enzymes, including HRP and AP), VENTANA iVIEW Detection System (biotinylated anti-species secondary antibodies and streptavidin-conjugated enzymes), VENTANA OptiView Detection System (OptiView) (anti-species secondary antibodies conjugated to haptens and anti-hapten tertiary antibodies conjugated to enzyme multimers), VENTANA Amplification Kit (unconjugated secondary antibodies that can be used with any of the previous VENTANA Detection Systems to amplify the number of enzymes at the site of primary antibody binding), VENTANA OptiView and the VENTANA DISCOVERY, DISCOVERY OmniMap, DISCOVERY UltraMap anti-hapten antibody, secondary antibody, chromogen, fluorophore, and dye kits, each of which is available from Ventana Medical Systems, Inc. (Tucson, Arizona), PowerVision and PowerVision+ IHC Detection Systems (secondary antibodies that polymerize directly with HRP or AP into compact polymers with high enzyme-to-antibody ratios), and the DAKO EnVision™+ System (enzyme-labeled polymers that bind to secondary antibodies).

Optionally, biomarker-stained slides can be counterstained to help identify morphologically relevant regions. Examples of counterstains include chromogenic nuclear counterstains such as hematoxylin (stains blue to purple), methylene blue (stains blue), toluidine blue (stains nuclei dark blue, polysaccharides pink to red), nuclear fast red (also called Kerneckrott stain, stains red) and methyl green (stains green); non-nucleogenic stains such as eosin (stains pink); 4',6-diamino-2-phenylindole (DAPI, stains blue), propidium iodide (stains red), Hoechst stain (stains blue), nuclear green DCS1 (stains green), nuclear yellow (Hoechst S769121, stains yellow at neutral pH and blue at acidic pH), DRAQ5 (stains red), DRAQ7 (stains red); fluorescent non-nuclear stains such as fluorophore-labeled phalloidin (stains filamentous actin, color depends on conjugated fluorophore).

III. Staining Assessment In one embodiment, a set of stained samples generated by the presently disclosed methods is used to determine the presence or absence of fusion proteins in patient samples. Typically, samples are obtained from a patient and prepared for analysis as described above. A portion of the sample (e.g., an initial tissue section from a biopsy of a tumor resection sample, or an initial slide prepared from a cytological sample of tumor cells (such as a cell smear (such as an endocervical smear), fine needle aspirate, isolated circulating tumor cells, etc.) is prepared and stained with a biomarker-specific reagent. The stained sample is then scored for the percentage of cell staining and/or the percentage of cells stained at or above a predefined threshold level. Preferably, the samples are fixed with formalin solution. In one embodiment, the threshold staining intensity is at least 1.5+, and the threshold level of tumor cells having the threshold staining intensity is in the range of 25%-75%. In one embodiment, the threshold staining intensity is at least 1.5+, and the threshold level of tumor cells having the threshold staining intensity is in the range of 50%-75%. In one embodiment, the threshold staining intensity is at least 1.5+ and the threshold level of tumor cells having the threshold staining intensity is 60%. In one embodiment, the threshold staining intensity is at least 1.5+ and the threshold level of tumor cells having the threshold staining intensity is 75%. In one embodiment, the threshold staining intensity is at least 2+ and the threshold level of tumor cells having the threshold staining intensity is in the range of 25%-75%. In one embodiment, the threshold staining intensity is at least 2+ and the threshold level of tumor cells having the threshold staining intensity is 50%. In one embodiment, the threshold staining intensity is at least 2+ and the threshold level of tumor cells having the threshold staining intensity is 60%. In one embodiment, the threshold staining intensity is at least 2+ and the threshold level of tumor cells having the threshold staining intensity is 75%.

In some cases, stained samples are stratified based on expression patterns and scored with a scoring methodology specific to the staining pattern. For example, if a fixed gradient is observed, a first scoring methodology is applied to samples with a cytoplasmic and/or membrane expression pattern and a second scoring methodology is applied to samples with nuclear cellular localization. For example, samples with a cytoplasmic and/or membrane staining pattern may be scored by determining whether the sample meets or exceeds a threshold level of tumor cells at or above a threshold staining intensity that is considered positive for the TRK fusion protein. In one embodiment, the threshold staining intensity is at least 1.5+ and the threshold level of tumor cells with the threshold staining intensity is in the range of 25%-75%. In one embodiment, the threshold staining intensity is at least 1.5+ and the threshold level of tumor cells with the threshold staining intensity is 50%. In one embodiment, the threshold staining intensity is at least 1.5+ and the threshold level of tumor cells with the threshold staining intensity is 60%. In one embodiment, the threshold staining intensity is at least 1.5+ and the threshold level of tumor cells with the threshold staining intensity is 75%. In one embodiment, the threshold staining intensity is at least 2+ and the threshold level of tumor cells having the threshold staining intensity is in the range of 25% to 75%. In one embodiment, the threshold staining intensity is at least 2+ and the threshold level of tumor cells having the threshold staining intensity is 50%. In one embodiment, the threshold staining intensity is at least 2+ and the threshold level of tumor cells having the threshold staining intensity is 60%. In one embodiment, the threshold staining intensity is at least 2+ and the threshold level of tumor cells having the threshold staining intensity is 75%.

As another example, samples with nuclear staining patterns can be scored by determining whether the minimum contiguous tumor cell area of the sample has a concentration of tumor cells that stain at or above a threshold expression level. In one embodiment, the threshold contiguous tumor cell area is at least 20 cells, the threshold staining intensity is any specific staining against background, and the threshold percentage of cells ranges from 25% to 75% or 25% to 80%. In another embodiment, the threshold contiguous tumor cell area is at least 20 cells, the threshold staining intensity is any specific staining against background, and the threshold percentage of cells is at least 75%. In another embodiment, the threshold contiguous tumor cell area is at least 20 cells, the threshold staining intensity is any specific staining against background, and the threshold percentage of cells is at least 80%. In one embodiment, the threshold contiguous tumor cell area is at least 20 cells, the threshold staining intensity is 0.5+ or above, and the threshold percentage of cells ranges from 25% to 75% or 25% to 80%. In another embodiment, the threshold adjacent tumor cell area is at least 20 cells, the threshold staining intensity is 0.5+ or greater, and the threshold percentage of cells is at least 75%. In another embodiment, the threshold adjacent tumor cell area is at least 20 cells, the threshold staining intensity is 0.5+ or greater, and the threshold percentage of cells is at least 80%. In one embodiment, the threshold adjacent tumor cell area is at least 50 cells, the threshold staining intensity is any specific staining against background, and the threshold percentage of cells ranges from 25%-75% or 25%-80%. In another embodiment, the threshold adjacent tumor cell area is at least 50 cells, the threshold staining intensity is any specific staining against background, and the threshold percentage of cells is at least 75%. In another embodiment, the threshold adjacent tumor cell area is at least 50 cells, the threshold staining intensity is any specific staining against background, and the threshold percentage of cells is at least 80%. In one embodiment, the threshold adjacent tumor cell area is at least 50 cells, the threshold staining intensity is 0.5+ or greater, and the threshold percentage of cells ranges from 25% to 75% or 25% to 80%. In another embodiment, the threshold adjacent tumor cell area is at least 50 cells, the threshold staining intensity is 0.5+ or greater, and the threshold percentage of cells is at least 75%. In another embodiment, the threshold adjacent tumor cell area is at least 50 cells, the threshold staining intensity is 0.5+ or greater, and the threshold percentage of cells is at least 80%.

VI. Clinical Applications In one embodiment, the assay described herein is used to characterize tumor samples from patients. For example, biopsy or resection samples are obtained, fixed, embedded in paraffin, sectioned and stained. The stained sections are scored as described above. In some embodiments, tumors with scores indicating the presence of fusion protein are characterized as "fusion positive", while tumors with scores not indicating the presence of fusion protein are characterized as "fusion negative". In other embodiments, cells in the intermediate category between "fusion positive" and "fusion negative" are characterized as "borderline". For example, if the scoring method is based on a threshold percentage of cells that stain at or above a threshold level, the percentage range below the threshold percentage is defined as "borderline", and all other samples with a percentage of cells that falls below the borderline range are considered "fusion negative". As another example, if the scoring method is based on a threshold percentage of cells staining at or above a threshold level within a threshold tumor cell area, the percentage range below the threshold tumor cell area is defined as "borderline" and all other samples with a percentage of cells that falls below the borderline range are considered "fusion negative."

In some embodiments, the assay is used as a screening test to identify patients eligible for a nucleic acid-based assay to confirm the presence of the fusion protein. For example, samples can be screened for the presence or absence of a fusion protein using an assay, and only samples characterized as fusion positive are subjected to a sequencing-based or PCR-based assay to confirm the presence and/or identity of the fusion detected in the assay. In other embodiments, the assay is a reflex test to confirm the presence and expression of the fusion protein identified by the nucleic acid-based assay. For example, samples can be screened for the presence or absence of an NTRK gene rearrangement using a sequencing-based or PCR-based assay, and only samples characterized as rearrangement positive by the sequencing-based or PCR-based assay are screened by the assay described herein to confirm the presence and/or expression of the fusion detected by the nucleic acid assay. In other embodiments, the characterization of the presence or absence of the fusion protein is based solely on the assay. In still other embodiments, samples characterized as "fusion negative" or "borderline" can be screened for the presence of an NTRK gene rearrangement by a sequencing-based or PCR-based assay, and cells characterized as "fusion positive" are not screened.

In some embodiments, the assay is used to select a therapy for a patient. For example, a patient with a tumor or sample characterized as "fusion positive" receives a targeted therapy directed against the wild-type counterpart, optionally in combination with a standard course of treatment for the tumor. Exemplary targeted therapies include those listed in Table 5.

Patients with tumors or samples characterized as fusion-negative receive standard treatment, which does not include targeted therapy against the wild-type counterpart.

VIII. EXAMPLES To evaluate whether a scoring algorithm could be developed that could predict the presence of Trk fusions across tumor types, IHC staining and analysis of over 3000 tissues across multiple indications was performed.

Formalin-fixed, paraffin-embedded (FFPE) normal and neoplastic tissues were obtained commercially. Tissues were screened with a prototype IHC assay using the pan-TRK antibody clone EPR17341, which included heat-induced epitope retrieval followed by antibody incubation and colorimetric development using OptiView DAB Detection (RTD, Tucson, AZ) on a BenchMark ULTRA automated slide stainer (RTD, Tucson, AZ).

A subset of cases, mostly with specific staining by pan-TRK (EPR17341) assay, were further evaluated by in situ hybridization (ISH) with NTRK1, 2, and 3 break-apart probes. The NTRK1, 2, and 3 break-apart oligo probes consisted of two pools of oligonucleotides (oligos) targeting genomic regions spanning the 5' and 3' ends of the NTRK1, 2, and 3 genes, respectively. DNP and fluorescein haptens were attached to the oligos in a basic intercalation configuration during synthesis using DNP-TEG phosphoramidite (Link Technologies Ltd, Bellshill, Lanarkshire) and 6-fluorescein phosphoramidite (Link Technologies Ltd, Bellshill, Lanarkshire). The synthetic oligos were purified using reverse-phase cartridges and mass spectrometry was performed to confirm removal of the truncated oligos.

The criteria used to define break-apart status for the ISH assay were determined based on evaluation of the mean break-apart rate in normal tissues and tumors without pan-TRK IHC expression. Tumors with a break-apart rate greater than 5 standard deviations above the mean were considered to represent true NTRK gene fusions.

Representative images of pan-TRK staining across various tumor types with various genotypes are shown in Figures 2A-2B. 9% of tumors (n=324) showed specific pan-TRK IHC staining, defined as >0% of tumor cell staining (range: 0%-54% for gastric and salivary gland cancers, respectively). When only specimens with IHC specific staining in ≥10% and ≥25% of tumor cells were considered, the overall percentage of cases with specific staining decreased to 5% (n=191) and 4% (n=133), respectively.

One hundred and sixty-four cases showing staining by IHC were available for ISH testing. Of these, 12 were found to harbor NTRK fusions (5 CRC, 2 melanoma, 2 papillary thyroid carcinoma, 2 salivary gland carcinoma, and 1 pancreatic carcinoma). The proportion of pan-TRK positive cases with various tumor cell staining percentages by tumor type, including the results of confirmatory testing by ISH, is shown in Table 6.

The intensity of staining and the percentage of tumor cells stained varied by tissue type, but cases with fusion tended to have higher intensity and higher percentage of tumor cells staining (see Figures 3A and 3B). Figures 3A and 3B show the distribution of ISH-confirmed samples by percentage of tumor cells staining (0-100%) and intensity of staining (0-3+). Circles represent fusion-negative cases and triangles represent fusion-positive cases. Colors represent different tumor histologies. Figure 3A shows the staining distribution of tumor types with ISH-confirmed fusion-positive cases in the dataset, and Figure 3B shows the staining distribution of neuroendocrine tumors where no fusion-positive cases were detected.

The results in terms of percentage of tumor cell staining by IHC for all tumors in the dataset are shown in Table 7.

12/164 (7%) of cases with IHC specific staining >0% of tumor cells showed the presence of NTRK fusion by ISH. 10/88 (11%) of cases with IHC specific staining >25% of tumor cells showed the presence of fusion by ISH (Table 6). Tumors showing fusion by ISH tended to stain with higher intensity and a higher percentage of tumor by pan-TRK IHC (upper right quadrant; Fig. 3A) than nonfusion tumors, with the exception of the neuroendocrine tumor (Fig. 3B). Using a higher definition of IHC specific positive staining (>25%), two fusion-positive melanomas were excluded from ISH testing (Table 6). These two tissue samples stained poorly by IHC and show evidence of a fixation gradient. Importantly, these tissues showed a high percentage (>90%) of highly intense nuclear staining in adjacent cells in areas that were not affected by fixation-related artifact.

The subcategorization of solid tumors based on IHC and ISH testing is shown in Figure 4.

Examples of observed staining patterns are shown in Figures 5 and 6. As shown in Figure 5, the staining patterns sometimes included aspects of membrane staining (A), cytoplasmic staining (A-C), and nuclear staining (C). Figure 7 shows that a fixation gradient occurred in some samples, which may affect the staining levels of some cells. It was noted that samples with nuclear localization were more sensitive to the fixation gradient than samples with cytoplasmic/membrane localization.

In summary, pan-TRK IHC staining varies in both intensity of staining and percentage of tumor cells depending on tumor type. Solid tumors with ISH-based fusions showed IHC staining at higher intensity and in a higher percentage of tumor cells than tumors with wild-type TRK protein expression, except for neuroendocrine and spindle cell tumors. In tumor types with a low prevalence of wild-type TRK protein expression (e.g., CRC and papillary thyroid carcinoma), pan-TRK IHC staining may help distinguish tumors with fusions. In contrast, in tumors with a high prevalence of wild-type TRK protein expression (e.g., neuroendocrine tumors, GIST), the distribution of percentage and intensity of IHC staining did not support a clear distinction between wild-type expression and fusions. According to Table 6, the number of specimens (out of a total of 164) subjected to ISH progressively decreased as the percentage of tumor cells staining by pan-TRK IHC increased, but this decrease did not impair the identification of tumors with NTRK gene fusions by ISH in well-preserved tissues. This metric, combined with the increasing intensity of IHC staining, appears to further highlight tumors with NTRK gene fusions by ISH testing.

Two melanomas and one pancreatic ductal adenocarcinoma did not conform to the above observations. The observed lower percentage of tumor cell staining was probably related to fixation artifact. Interestingly, however, small areas of adjacent viable tumor showed a high percentage of nuclear staining at high intensity.

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Claims (15)

1. A method for detecting a TrkA, TrkB, or TrkC fusion protein in a non-neuroendocrine tumor sample, comprising:
- affinity histochemical staining of said sample with a biomarker-specific reagent that specifically binds to one or more of the following : an amino acid sequence comprising, or consisting of, residues 363-760 of SEQ ID NO:1; an amino acid sequence comprising, or consisting of , residues 646-838 of SEQ ID NO:2; or an amino acid sequence comprising, or consisting of, residues 718-839 of SEQ ID NO:3;
- detecting a nuclear staining of said sample ;
- scoring the sample as positive for a fusion protein involving TrkA, TrkB, or TrkC if the detected nuclear staining has a staining pattern that is equal to or greater than a threshold percentage of tumor cells within a threshold contiguous tumor cell area that are specifically stained above a threshold staining intensity;
Including,
the threshold contiguous tumor cell region is at least 20 contiguous cells;
method. The method of claim 1 , wherein the threshold percentage is in the range of 25 % to 80 %, and the threshold staining intensity is any specific staining above background. The method of claim 1 , wherein the threshold percentage is between 25% and 75% and the first specific staining intensity is greater than 0.5+ . The method of any one of claims 1 to 3 , wherein the threshold percentage is in the range of 20% to 90%, and the threshold staining intensity is any specific staining above background. 5. The method of claim 1 , wherein the threshold contiguous tumor cell area is at least 50 cells , the threshold staining intensity is 0.5+ or greater, and the threshold percentage is in the range of 25% to 75%. 6. The method of any one of claims 1-5 , wherein the biomarker-specific reagent is an antibody, and the antibody is immunospecific for an epitope located in the amino acid sequence consisting of amino acids 816-838 of SEQ ID NO:2. The method of claim 6 , wherein the antibody is monoclonal antibody clone EPR17341. The affinity histochemical staining
(a) subjecting the sample to a heat-induced epitope retrieval process; and (b) contacting the sample with a set of biomarker-specific and detection reagents to deposit a bright field dye proximate to any biomarker-specific reagents bound to the sample.
The method according to any one of claims 1 to 7 , comprising: 10. The method of claim 8, wherein: (b1) the biomarker-specific reagent is an antibody that is specifically immunoreactive with each of the amino acid sequence consisting of residues 363-760 of SEQ ID NO:1; the amino acid sequence consisting of residues 646-838 of SEQ ID NO:2; and the amino acid sequence consisting of residues 718-839 of SEQ ID NO:3; and (b2) the set of detection reagents comprises a secondary antibody immunoreactive with the antibody of ( b1 ), a tertiary antibody immunoreactive with the secondary antibody and conjugated to an enzyme, and a set of reagents reactive with the enzyme that result in deposition of a bright field dye into the sample. The method of claim 9 , wherein the secondary antibody is haptenized and the tertiary antibody is an anti-hapten antibody. 11. A method for detecting NTRK rearrangements in a non-neuroendocrine tumor sample, comprising detecting the presence of a Trk fusion protein in the sample according to the method of any one of claims 1 to 10 , and if the sample scores positive for a fusion protein involving TrkA, TrkB, or TrkC, screening the sample for NTRK rearrangements by sequencing, reverse transcription polymerase chain reaction, or in situ hybridization. 12. The method of claim 11, further comprising identifying samples as either Trk fusion positive or Trk fusion negative, and a screening step comprising screening samples that are Trk fusion negative for NTRK rearrangements by sequencing, reverse transcription polymerase chain reaction, or in situ hybridization . 12. The method of claim 11, further comprising identifying samples as either Trk fusion positive, Trk fusion borderline (Equivocal) or Trk fusion negative, and a screening step comprising screening samples that are Trk fusion borderline for NTRK rearrangements by sequencing, reverse transcription polymerase chain reaction, or in situ hybridization. selecting the patient for a Trk-directed therapy if the non-neuroendocrine tumor sample scores positive for a TRK fusion protein involving TrkA, TrkB, or TrkC, or if an NTRK rearrangement is detected;
The threshold level of adjacent tumor cells is at least 20 adjacent cells, the threshold percentage is in the range of 20% to 90%, and the threshold staining intensity is any specific staining above background.
14. The method according to claim 12 or 13 . A method for selecting a patient to receive a Trk-directed therapy, comprising detecting the presence of a Trk fusion protein in a non-neuroendocrine tumor sample according to the method of any one of claims 1 to 10 , and selecting said patient to receive said therapy if said sample scores positive for a fusion protein involving TrkA, TrkB or TrkC, or if an NTRK rearrangement is detected,
The threshold level of adjacent tumor cells is at least 20 adjacent cells, the threshold percentage ranges from 20% to 90%, and the threshold staining intensity is any specific staining above background.
method.

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